Nature generates functional molecules through integrated cycles of diversification, selection, and amplification. While researchers have demonstrated the power of adapting this approach in the laboratory to generate proteins with desired binding or catalytic properties, the scope of protein molecular evolution is currently limited by the small number of strategies to screen or select for proteins with desired properties. Selections performed inside the living cell, in particular, are rare despite the significant advantages of in vivo selections over in vitro selections or screens. Moreover, while several proteins have been evolved to accept new substrates, the result of a selection or screen for new substrate tolerance is often an enzyme with broadened, rather than altered specificity. Our work seeks to address these existing limitations of protein molecular evolution. We propose the development of coupled positive and negative in vivo selections for the evolution of three classes of enzymes that manipulate biological macromolecules in powerful ways: recombinases, homing endonucleases, and inteins. The successful implementation of these methods will expand our understanding of these three important classes of enzymes and will validate the potential of coupled in vivo positive and negative selections to generate enzymes with truly altered, rather than simply broadened, specificities and activities. In addition, the successful evolution of each of these enzymes towards new activities may produce powerful research tools such as ligand-activated protein splicing domains and enzymes that specifically cleave or recombine DNA at sequences chosen by the researcher. Proteins evolved in this manner may also represent novel therapeutic approaches to protecting cells from viral infection and to manipulating virtually any protein-mediated process with a small molecule.